TRAF2-associated protein kinase and assays

ABSTRACT

The invention provides methods and compositions relating to a novel human tumor necrosis factor receptor associated factor number two associated kinase protein. The invention provides hybridization probes and primers capable of hybridizing with the disclosed gene, nucleic acids encoding the kinase, methods of making the kinase proteins, and methods of using the compositions in diagnosis and drug screening.

This is a divisional application of U.S. Ser. No. 08/677,862, filed Jul.10, 1996, allowed.

INTRODUCTION

1. Field of the Invention

The field of this invention is a class of human proteins involved ingene transcription.

2. Background

Nuclear factor κB (NF-κB) is a homo- or heterodimer of members of theRel family of transcriptional activators that is involved in theinducible expression of a wide variety of important cellular genesincluding numerous cytokines, cytokine receptors, majorhistocompatibility antigens, serum amyloid A protein, etc. as well asmany viral genes including genes of HIV, SV40, cytomegalovirus, etc.Several tumor necrosis factor receptor-associated factor (TRAF) proteinshave been identified and shown to be involved in the signaling ofvarious cellular responses including cytotoxicity, anti-viral activity,immuno-regulatory activities and the transcriptional regulation of anumber of genes.

Accordingly, the ability to exogenously modulate the activity of NF-κBand/or TRAF proteins would yield therapeutic application for numerousclinical indications. In addition, components of such pathways wouldprovide valuable target reagents for automated, cost-effective, highthroughput drug screening assays and hence would have immediateapplication in domestic and international pharmaceutical andbiotechnology drug development programs. The present invention providesnovel TRAF-2 associated kinase proteins which regulate TRAF-2 function,their use, e.g. in drug screens, and nucleic acids encoding the same.

3. Relevant Literature

Kentrup et al. (1996) J. Biol. Chem 271, 3488-3495, report the existenceof Dyrk, a rat protein kinase with sequence similarity with the humankinase disclosed herein.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to a novelhuman TRAF2-associated protein kinase and gene. The subject kinaseproteins comprise a functional domain of SEQ ID NO:2 distinguishable(e.g. in terms of sequence or function, such a binding specificity) fromrodent homologs of the kinase. For example, SEQ ID NO:2, residues 1-158,159-479 and 480-763 provide human-specific C, kinase and N domains,respectively. The invention also provides isolated hybridization probesand primers capable of specifically hybridizing with or amplifying thedisclosed human kinase protein gene (SEQ ID NO: 1), nucleic acidsencoding the subject proteins, methods of making the subject proteinsand nucleic acids, and methods of using the subject compositions indiagnosis (e.g. genetic hybridization screens for gene mutations), andin the biopharmaceutical industry (e.g. reagents for screening chemicallibraries for lead compounds for a pharmacological agent useful in thediagnosis or treatment of disease associated with immune regulation).

DESCRIPTION OF THE DRAWING

FIG. 1. Deletion mutant analysis of kinase proteins for TRAF2 binding.

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of a natural cDNA encoding a novel humanTRAF2-associated protein kinase is shown as SEQ ID NO: 1 and the fullconceptual translate shown as SEQ ID NO:2. The kinase proteins of theinvention include incomplete translates of SEQ ID NO: 1 and deletionmutants of SEQ ID NO: 2, which translates and deletions mutants haveamino acid sequence and binding specificity or function different fromrodent homologs of the protein. For example, the domain bound byresidues 159 (Tyr) through 479 (Phe) of SEQ ID NO:2 defines an activekinase domain which may be used, independently or joined to otherdomains, in the subject methods; see FIG. 1. Also, an internal domainwithin residues 159-598 of SEQ ID NO:2 includes a TRAF-2 binding domain.This domain finds use in methods involving kinase-TRAF-2 complexes andmay be used independently as a regulator of TRAF-2 activity, as areagent in kinase complex formation assays, etc.

The binding or function specificity of the subject proteins necessarilydistinguishes, qualitatively and/or quantitatively, rodent homologs(e.g. the rat Dyrk gene product). This specificity is especiallyimportant for screens for lead pharmaceuticals (below). Binding orfunction specificity may be determined by convenient in vitro,cell-based, or in vivo assays. Preferred proteins have kinase activity(e.g. autophosphorylate), specifically bind TRAF2 or modulate NF-κBactivation. Such activity or function may be demonstrated in in vitrobinding assays, in cell culture (e.g. cell transfections) or in animals(e.g. in vivo gene therapy, transgenics). Generally, binding specificityis shown by kinase activity, by binding equilibrium constants (usuallyat least about 10⁷ M⁻¹, preferably at least about 10⁸ M⁻¹, morepreferably at least about 10⁹ M⁻¹) with natural binding targets such ashTRAF2 or nonnatural targets such as specific antibodies, by the abilityof the subject protein to elicit a specific antibody in a rodent orrabbit (i.e. an antibody which distinguishes the subject proteins fromrodent homologs), etc.

The claimed proteins are isolated or pure and are typicallyrecombinantly produced. An "isolated" protein is unaccompanied by atleast some of the material with which it is associated in its naturalstate, preferably constituting at least about 0.5%, and more preferablyat least about 5% by weight of the total protein in a given sample and apure protein constitutes at least about 90%, and preferably at leastabout 99% by weight of the total protein in a given sample. A widevariety of molecular and biochemical methods are available forgenerating, expressing and purifying the subject compositions, see e.g.Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold SpringHarbor Laboratory), Current Protocols in Molecular Biology (Eds.Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or thatare otherwise known in the art.

The invention provide binding agents specific to the subject kinaseproteins including substrates, agonists, antagonists, naturalintracellular binding targets, etc., methods of identifying and makingsuch agents, and their use in diagnosis, therapy and pharmaceuticaldevelopment. For example, specific binding agents are useful in avariety of diagnostic and therapeutic applications, especially wheredisease or disease prognosis is associated with improper utilization ofa pathway involving the subject proteins, e.g. NF-κB activation. Novelspecific binding agents include specific antibodies and other naturalintracellular binding agents identified with assays such as one-, two-and three-hybrid screens, non-natural intracellular binding agentsidentified in screens of chemical libraries such as described below,etc.

The invention also provides nucleic acids encoding the subject proteins,which nucleic acids may be part of expression vectors and may beincorporated into recombinant cells for expression and screening,transgenic animals for functional studies (e.g. the efficacy ofcandidate drugs for disease associated with signal transduction mediatedby the subject kinase proteins), etc., and nucleic acid hybridizationprobes and replication/amplification primers having a cDNA specificsequence contained in SEQ ID NO: 1 and sufficient to effect specifichybridization thereto (i.e. specifically hybridize with SEQ ID NO: 1 inthe presence of natural cDNAs encoding rodent homologs, eg. rat DyrkcDNA (Kentrup et al., 1996, supra).

The subject nucleic acids are isolated, i.e. unaccompanied by at leastsome of the material with which it is associated in its natural state,preferably constituting at least about 0.5%, preferably at least about5% by weight of total nucleic acid present in a given fraction, andusually recombinant, meaning they comprise a sequence joined to anucleotide other than that which it is joined to on a naturalchromosome. Nucleic acids comprising the nucleotide sequence of SEQ IDNO: 1 or fragments thereof, contain such sequence or fragment at aterminus, immediately flanked by a sequence other than that which it isjoined to on a natural chromosome, or flanked by a native flankingregion fewer than 10 kb, preferably fewer than 2 kb, which isimmediately flanked by a sequence other than that which it is joined toon a natural chromosome. The subject nucleic acids find a wide varietyof applications including use as translatable transcripts, hybridizationprobes, PCR primers, diagnostic nucleic acids, etc.; use in detectingthe presence of the subject genes and gene transcripts and in detectingor amplifying nucleic acids encoding additional homologs and structuralanalogs. In diagnosis, the hybridization probes and/or primers find usein identifying wild-type and mutant alleles in clinical and laboratorysamples. Mutant alleles are used to generate reagents e.g.allele-specific oligonucleotides (ASO), for high-throughput clinicaldiagnoses.

The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of cellularfunction modulated by the disclosed protein kinases. Generally, thesescreening methods involve assaying for compounds which modulateinteraction with a natural binding target. A wide variety of assays forbinding agents are provided including labeled in vitro kinase assays,protein-protein binding assays, immunoassays, cell based assays, etc.The methods are amenable to automated, cost-effective high throughputscreening of chemical libraries for lead compounds. Identified reagentsfind use in the pharmaceutical industries for animal and human trials;for example, the reagents may be derivatized and rescreened in in vitroand in vivo assays to optimize activity and minimize toxicity forpharmaceutical development. Target indications may include infection,genetic disease, cell growth and regulatory disfunction, such asneoplasia, inflammation, hypersensitivity, etc.

In vitro binding assays employ a mixture of components including asubject protein kinase, which may be part of a fusion product withanother peptide or polypeptide, e.g. a polypeptide that is capable ofproviding or enhancing protein-protein binding, stability under assayconditions, or a tag for detection or anchoring, etc. The assay mixturescomprise a natural intracellular specific-binding target, e.g. asubstrate, such as TRAF2. A pseudosubstrate may be used or modified(e.g. A to S/T substitutions) to generate effective substrates for usein the subject kinase assays. While native binding targets may be used,it is frequently preferred to use portions (e.g. peptides, nucleic acidfragments) or analogs thereof so long as the portion or analog providesbinding affinity and avidity to the subject protein kinase convenientlymeasurable in the assay. The assay mixture also comprises a candidatepharmacological agent. Candidate agents encompass numerous chemicalclasses, though typically they are organic compounds; preferably smallorganic compounds and are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. A variety ofother reagents may also be included in the mixture. These includereagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc. which may be used to facilitate optimal binding and/orreduce non-specific or background interactions, etc. Also, reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for thepresence of the candidate pharmacological agent, the kinase proteinspecifically binds the binding target, with a reference bindingaffinity. The mixture components can be added in any order that providesfor the requisite bindings and incubations may be performed at anytemperature which facilitates optimal binding, typically between 4° and40° C., more commonly between 15° and 40° C. Incubation periods arelikewise selected for optimal binding but also minimized to facilitaterapid, high-throughput screening, and are typically between 0.1 and 10hours, preferably less than 5 hours, more preferably less than 2 hours.

After incubation, the agent-biased binding between the kinase proteinand one or more binding targets is detected by any convenient way. Forcell-free binding type assays, a separation step is often used toseparate bound from unbound components. Separation may be effected byprecipitation (e.g. TCA precipitation, immunoprecipitation, etc.),immobilization (e.g on a solid substrate), etc., followed by washing by,for examples, membrane filtration (e.g. Whatman's P-81 ion exchangepaper, Polyfiltronic's hydrophobic GFC membrane, etc.), gelchromatography (e.g. gel filtration, affinity, etc.). For kinase assays,binding is detected by a change in the kinase-induced phosphorylation ofthe substrate.

Detection may be effected in any convenient way. For cell-free bindingassays, one of the componentually comprises or is coupled to a label. Awide variety of labels may be employed--essentially any label thatprovides for detection of bound protein. The label may provide fordirect detection as radioactivity, luminescence, optical or electrondensity, etc. or indirect detection such as an epitope tag, an enzyme,etc. A variety of methods may be used to detect the label depending onthe nature of the label and other assay components. For example, thelabel may be detected bound to the solid substrate or a portion of thebound complex containing the label may be separated from the solidsubstrate, and thereafter the label detected. Labels may be directlydetected through optical or electron density, radiative emissions,nonradiative energy transfers, etc. or indirectly detected with antibodyconjugates, etc. For example, in the case of radioactive labels,emissions may be detected directly, e.g. with particle counters orindirectly, e.g. with scintillation cocktails and counters.

A difference in the binding affinity of the kinase protein to the targetin the absence of the agent as compared with the binding affinity in thepresence of the agent indicates that the agent modulates the binding ofthe kinase protein to the binding target. Analogously, in the cell-basedtranscription assay also described below, a difference in thetranscriptional induction in the presence and absence of an agentindicates the agent modulates transcription induced by the subjectkinase protein. A difference, as used herein, is statisticallysignificant and preferably represents at least a 50%, more preferably atleast a 90% difference.

The following experiments and examples are offered by way ofillustration and not by way of limitation.

EXPERIMENTAL

A human kinase protein was initially identified in immunoprecipitates ofTRAF2. Coprecipitating proteins were purified and subject to peptidesequencing. The resultant sequence data were used to designoligonucleotide probe and primers to isolate human cDNA clones.Identification was confirmed by overexpressing a full-length myc-taggedkinase-encoding cDNA in human 293 cells cotransfected with FLAG-taggedTRAF2 and immunoprecipitating the lysates with anti-FLAG then westernblot analysis with anti-myc. A yeast two-hybrid system was also used toconfirm TRAF2 binding and for deletion mutagenesis analysis of kinase.These experiments revealed that residues 1-763, residues 1-598 andresidues 159-763 are each sufficient to mediate TRAF2 binding, whileresidues 1-567 is not. Human kinase peptides derived from the 567-598are able to inhibit kinase-TRAF2 binding. Sequence analysis furtherdefine a kinase domain of residues 159-479. Recombinant kinase wasprepared by over-expressing GST fusion proteins in E. coli andbaculavirus expression systems.

EXAMPLES 1. Protocol for Autophosphorylation Assay

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

kinase: 10⁻⁸ -10⁻⁵ M biotinylated kinase (SEQ ID NO:2) at 20 μg/ml inPBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH 7.4,0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktailof protease inhibitors.

[³² P]γ-ATP 10× stock: 2×10⁻⁵ M cold ATP with 100 μCi [³² P]γ-ATP. Placein the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 40 μl biotinylated kinase (0.1-10 pmoles/40 ul in assay buffer)

Add 10 μl compound or extract.

Add 10 μl [³² P]γ-ATP 10× stock.

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. cold ATP at 80% inhibition.

2. Protocol for Kinase Protein--hTRAF2 Complex Formation Assay

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH 7.6,0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/mlBSA, cocktail of protease inhibitors.

³³ P kinase protein 10× stock: 10⁻⁸ -10⁻⁶ M "cold" kinase protein (SEQID NO:2, residues 159-598) supplemented with 200,000-250,000 cpm oflabeled kinase protein (Beckman counter). Place in the 4° C. microfridgeduring screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

hTRAF2: 10⁻⁸ -10⁻⁵ M biotinylated hTRAF2 in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³ P-kinase protein (20,000-25,000 cpm/0.1-10 pmoles/well=10⁻⁹-10⁻⁷ M final concentration).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μl biotinylated hTRAF2 (0.1-10 pmoles/40 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated hTRAF2) at 80% inhibition.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3218 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATGGAGCTCC ACCGCGGTGG CGGCCGCTCT AGAACTAGTG GATCCCCCAT AG -             #TTTTGCCG     60                                                                 - - CTGGACTCTT CCCTCCCTTC CCCCACCCCA TCAGGATGAT ATGAGACTTG AA -            #AGAAGACG    120                                                                 - - ATGCATACAG GAGGAGAGAC TTCAGCATGC AAACCTTCAT CTGTTCGGCT TG -            #CACCGTCA    180                                                                 - - TTTTCATTCC ATGCTGCTGG CCTTCAGATG GCTGGACAGA TGCCCCATTC AC -            #ATCAGTAC    240                                                                 - - AGTGACCGTC GCCAGCCAAA CATAAGTGAC CAACAGGTTT CTGCCTTATC AT -            #ATTCTGAC    300                                                                 - - CAGATTCAGC AACCTCTAAC TAACCAGGTG ATGCCTGATA TTGTCATGTT AC -            #AGAGGCGG    360                                                                 - - ATGCCCCAAA CCTTCCGTGA CCCAGCAACT GCTCCCCTGA GAAAACTTTC TG -            #TTGACTTG    420                                                                 - - ATCAAAACAT ACAAGCATAT TAATGAGGTT TACTATGCAA AAAAGAAGCG AA -            #GACACCAA    480                                                                 - - CAGGGCCAGG GAGACGATTC TAGTCATAAG AAGGAACGGA AGGTTTACAA TG -            #ATGGTTAT    540                                                                 - - GATGATGATA ACTATGATTA TATTGTAAAA AACGGAGAAA AGTGGATGGA TC -            #GTTACGAA    600                                                                 - - ATTGACTCCT TGATAGGCAA AGGTTCCTTT GGACAGGTTG TAAAGGCATA TG -            #ATCGTGTG    660                                                                 - - GAGCAAGAAT GGGTTGCCAT TAAAATAATA AAGAACAAGA AGGCTTTTCT GA -            #ATCAAGCA    720                                                                 - - CAGATAGAAG TGCGACTTCT TGAGCTCATG AACAAACATG ACACTGAAAT GA -            #AATACTAC    780                                                                 - - ATAGTGCATT TGAAACGCCA CTTTATGTTT CGAAACCATC TCTGTTTAGT TT -            #TTGAAATG    840                                                                 - - CTGTCCTACA ACCTCTATGA CTTGCTGAGA AACACCAATT TCCGAGGGGT CT -            #CTTTGAAC    900                                                                 - - CTAACACGAA AGTTTGCGCA ACAGATGTGC ACTGCACTGC TTTTCCTTGC GA -            #CTCCAGAA    960                                                                 - - CTTAGTATCA TTCACTGTGA TCTAAAACCT GAAAATATCC TTCTTTGTAA CC -            #CCAAACGC   1020                                                                 - - AGTGCAATCA AGATAGTTGA CTTTGGCAGT TCTTGTCAGT TGGGGCAGAG GA -            #TATACCAG   1080                                                                 - - TATATTCAGA GTCGCTTTTA TCGGTCTCCA GAGGTGCTAC TGGGAATGCC TT -            #ATGACCTT   1140                                                                 - - GCCATTGATA TGTGGTCCCT CGGGTGTATT TTGGTTGAAA TGCACACTGG AG -            #AACCTCTG   1200                                                                 - - TTCAGTGGTG CCAATGAGGT AGATCAGATG AATAAAATAG TGGAAGTTCT GG -            #GTATTCCA   1260                                                                 - - CCTGCTCATA TTCTTGACCA AGCACCAAAA GCAAGAAAGT TCTTTGAGAA GT -            #TGCCAGAT   1320                                                                 - - GGCACTTGGA ACTTAAAGAA GACCAAAGAT GGAAAACGGG AGTACAAACC AC -            #CAGGAACC   1380                                                                 - - CGTAAACTTC ATAACATTCT TGGAGTGGAA ACAGGAGGAC CTGGTGGGCG AC -            #GTGCTGGG   1440                                                                 - - GAGTCAGGTC ATACGGTCGC TGACTACTTG AAGTTCAAAG ACCTCATTTT AA -            #GGATGCTT   1500                                                                 - - GATTATGACC CCAAAACTCG AATTCAACCT TATTATGCTC TGCAGCACAG TT -            #TCTTCAAG   1560                                                                 - - AAAACAGCTG ATGAAGGTAC AAATACAAGT AATAGTGTAT CTACAAGCCC CG -            #CCATGGAG   1620                                                                 - - CAGTCTCAGT CTTCGGGCAC CACCTCCAGT ACATCGTCAA GCTCAGGTGG CT -            #CATCGGGG   1680                                                                 - - ACAAGCAACA GTGGGAGAGC CCGGTCGGAT CCGACGCACC AGCATCGGCA CA -            #GTGGTGGG   1740                                                                 - - CACTTCACAG CTGCCGTGCA GGCCATGGAC TGCGAGACAC ACAGTCCCCA GG -            #TGCGTCAG   1800                                                                 - - CAATTTCCTG CTCCTCTTGG TTGGTCAGGC ACTGAAGCTC CTACACAGGT CA -            #CTGTTGAA   1860                                                                 - - ACTCATCCTG TTCAAGAAAC AACCTTTCAT GTAGCCCCTC AACAGAATGC AT -            #TGCATCAT   1920                                                                 - - CACCATGGTA ACAGTTCCCA TCACCATCAC CACCACCACC ACCATCACCA CC -            #ACCATGGA   1980                                                                 - - CAACAAGCCT TGGGTAACCG GACCAGGCCA AGGGTCTACA ATTCTCCAAC GA -            #ATAGCTCC   2040                                                                 - - TCTACCCAAG ATTCTATGGA GGTTGGCCAC AGTCACCACT CCATGACATC CC -            #TGTCTTCC   2100                                                                 - - TCAACGACTT CTTCCTCGAC ATCTTCCTCC TCTACTGGTA ACCAAGGCAA TC -            #AGGCCTAC   2160                                                                 - - CAGAATCGCC CAGTGGCTGC TAATACCTTG GACTTTGGAC AGAATGGAGC TA -            #TGGACGTT   2220                                                                 - - AATTTGACCG TCTACTCCAA TCCCCGCCAA GAGACTGGCA TAGCTGGACA TC -            #CAACATAC   2280                                                                 - - CAATTTTCTG CTAATACAGG TCCTGCACAT TACATGACTG AAGGACATCT GA -            #CAATGAGG   2340                                                                 - - CAAGGGGCTG ATAGAGAAGA GTCCCCCATG ACAGGAGTTT GTGTGCAACA GA -            #GTCCTGTA   2400                                                                 - - GCTAGCTCGT GACTACATTG AAACTTGAGT TTGTTTCTTG TGTGTTTTTA TA -            #GAAGTGGT   2460                                                                 - - GTTTTTTTTC CAAAAACAAA GTGCAAAGCT GCTTGAATCA GGAGGAGATT AA -            #CACACTGA   2520                                                                 - - ACCGCTACAA GAGGGCAAAG CTGATTTTTT TTTTAACTTG AAAAGATTGC AA -            #AGGGACAT   2580                                                                 - - TGAAGTGTTT AAAAGAGCCA TGTCCAAACC CATCTTCATG GATAGCTCAG AG -            #GTATCCTC   2640                                                                 - - TTTTTGCTCC CCCATTTTAA CTTGCCACAT CCCAGTCACA GTGGGGTTTT TT -            #TGTCTTTC   2700                                                                 - - TATTCAGCAA AAGTTAATAT TCAGATGTTG GTCTTGGTCA TTTGCCAACT AA -            #TTTTAAAG   2760                                                                 - - TAAAAGGCAC TGCACATAAT TTGCATAAAG GGCCCCATGA GGGTGTTTTT TT -            #TTTTTCTT   2820                                                                 - - TTTGTCCCCC CCATCCCCCT TTTTTTTTGT TTTGTTCTGT TTTGTTTTGG GT -            #GGGAGGGT   2880                                                                 - - GGGAAATTTG GGTTTTTAAG TCCTCTAAAC ACACTTGGGC ACGGAAATGC AG -            #TACTGTAA   2940                                                                 - - GGAANANGGA CCTCCAGCTT CCACAAACAC CATCTTCAGC TGTATGAAAG GG -            #ACGGTTGT   3000                                                                 - - GGTGAAGTTT GTCAGGCACA GTAAGCATGC TGAGTGGCGG GGATCAGAAC TC -            #TCCTATCT   3060                                                                 - - GAACCTACTG AGGANCAAAG CAGCAATTAC ATGGATCCTG TGGCCNCCCC GT -            #TGCAAAGC   3120                                                                 - - CCAGGAANAN AAGATGNACN TGACTGGTCT CCTAACCAAG TGCNCTGAAA AC -            #CATCAACG   3180                                                                 - - GTCCGTCCTT GGCANTCCTG GGGAGTCTAA TTTGTGNC      - #                      - #   3218                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 763 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met His Thr Gly Gly Glu Thr Ser Ala Cys Ly - #s Pro Ser Ser Val Arg      1               5   - #                10  - #                15               - - Leu Ala Pro Ser Phe Ser Phe His Ala Ala Gl - #y Leu Gln Met Ala Gly                  20      - #            25      - #            30                   - - Gln Met Pro His Ser His Gln Tyr Ser Asp Ar - #g Arg Gln Pro Asn Ile              35          - #        40          - #        45                       - - Ser Asp Gln Gln Val Ser Ala Leu Ser Tyr Se - #r Asp Gln Ile Gln Gln          50              - #    55              - #    60                           - - Pro Leu Thr Asn Gln Val Met Pro Asp Ile Va - #l Met Leu Gln Arg Arg      65                  - #70                  - #75                  - #80        - - Met Pro Gln Thr Phe Arg Asp Pro Ala Thr Al - #a Pro Leu Arg Lys Leu                      85  - #                90  - #                95               - - Ser Val Asp Leu Ile Lys Thr Tyr Lys His Il - #e Asn Glu Val Tyr Tyr                  100      - #           105      - #           110                  - - Ala Lys Lys Lys Arg Arg His Gln Gln Gly Gl - #n Gly Asp Asp Ser Ser              115          - #       120          - #       125                      - - His Lys Lys Glu Arg Lys Val Tyr Asn Asp Gl - #y Tyr Asp Asp Asp Asn          130              - #   135              - #   140                          - - Tyr Asp Tyr Ile Val Lys Asn Gly Glu Lys Tr - #p Met Asp Arg Tyr Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Asp Ser Leu Ile Gly Lys Gly Ser Phe Gl - #y Gln Val Val Lys        Ala                                                                                             165  - #               170  - #               175             - - Tyr Asp Arg Val Glu Gln Glu Trp Val Ala Il - #e Lys Ile Ile Lys Asn                  180      - #           185      - #           190                  - - Lys Lys Ala Phe Leu Asn Gln Ala Gln Ile Gl - #u Val Arg Leu Leu Glu              195          - #       200          - #       205                      - - Leu Met Asn Lys His Asp Thr Glu Met Lys Ty - #r Tyr Ile Val His Leu          210              - #   215              - #   220                          - - Lys Arg His Phe Met Phe Arg Asn His Leu Cy - #s Leu Val Phe Glu Met      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Ser Tyr Asn Leu Tyr Asp Leu Leu Arg As - #n Thr Asn Phe Arg        Gly                                                                                             245  - #               250  - #               255             - - Val Ser Leu Asn Leu Thr Arg Lys Phe Ala Gl - #n Gln Met Cys Thr Ala                  260      - #           265      - #           270                  - - Leu Leu Phe Leu Ala Thr Pro Glu Leu Ser Il - #e Ile His Cys Asp Leu              275          - #       280          - #       285                      - - Lys Pro Glu Asn Ile Leu Leu Cys Asn Pro Ly - #s Arg Ser Ala Ile Lys          290              - #   295              - #   300                          - - Ile Val Asp Phe Gly Ser Ser Cys Gln Leu Gl - #y Gln Arg Ile Tyr Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Tyr Ile Gln Ser Arg Phe Tyr Arg Ser Pro Gl - #u Val Leu Leu Gly        Met                                                                                             325  - #               330  - #               335             - - Pro Tyr Asp Leu Ala Ile Asp Met Trp Ser Le - #u Gly Cys Ile Leu Val                  340      - #           345      - #           350                  - - Glu Met His Thr Gly Glu Pro Leu Phe Ser Gl - #y Ala Asn Glu Val Asp              355          - #       360          - #       365                      - - Gln Met Asn Lys Ile Val Glu Val Leu Gly Il - #e Pro Pro Ala His Ile          370              - #   375              - #   380                          - - Leu Asp Gln Ala Pro Lys Ala Arg Lys Phe Ph - #e Glu Lys Leu Pro Asp      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Thr Trp Asn Leu Lys Lys Thr Lys Asp Gl - #y Lys Arg Glu Tyr        Lys                                                                                             405  - #               410  - #               415             - - Pro Pro Gly Thr Arg Lys Leu His Asn Ile Le - #u Gly Val Glu Thr Gly                  420      - #           425      - #           430                  - - Gly Pro Gly Gly Arg Arg Ala Gly Glu Ser Gl - #y His Thr Val Ala Asp              435          - #       440          - #       445                      - - Tyr Leu Lys Phe Lys Asp Leu Ile Leu Arg Me - #t Leu Asp Tyr Asp Pro          450              - #   455              - #   460                          - - Lys Thr Arg Ile Gln Pro Tyr Tyr Ala Leu Gl - #n His Ser Phe Phe Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Lys Thr Ala Asp Glu Gly Thr Asn Thr Ser As - #n Ser Val Ser Thr        Ser                                                                                             485  - #               490  - #               495             - - Pro Ala Met Glu Gln Ser Gln Ser Ser Gly Th - #r Thr Ser Ser Thr Ser                  500      - #           505      - #           510                  - - Ser Ser Ser Gly Gly Ser Ser Gly Thr Ser As - #n Ser Gly Arg Ala Arg              515          - #       520          - #       525                      - - Ser Asp Pro Thr His Gln His Arg His Ser Gl - #y Gly His Phe Thr Ala          530              - #   535              - #   540                          - - Ala Val Gln Ala Met Asp Cys Glu Thr His Se - #r Pro Gln Val Arg Gln      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Gln Phe Pro Ala Pro Leu Gly Trp Ser Gly Th - #r Glu Ala Pro Thr        Gln                                                                                             565  - #               570  - #               575             - - Val Thr Val Glu Thr His Pro Val Gln Glu Th - #r Thr Phe His Val Ala                  580      - #           585      - #           590                  - - Pro Gln Gln Asn Ala Leu His His His His Gl - #y Asn Ser Ser His His              595          - #       600          - #       605                      - - His His His His His His His His His His Hi - #s Gly Gln Gln Ala Leu          610              - #   615              - #   620                          - - Gly Asn Arg Thr Arg Pro Arg Val Tyr Asn Se - #r Pro Thr Asn Ser Ser      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Ser Thr Gln Asp Ser Met Glu Val Gly His Se - #r His His Ser Met        Thr                                                                                             645  - #               650  - #               655             - - Ser Leu Ser Ser Ser Thr Thr Ser Ser Ser Th - #r Ser Ser Ser Ser Thr                  660      - #           665      - #           670                  - - Gly Asn Gln Gly Asn Gln Ala Tyr Gln Asn Ar - #g Pro Val Ala Ala Asn              675          - #       680          - #       685                      - - Thr Leu Asp Phe Gly Gln Asn Gly Ala Met As - #p Val Asn Leu Thr Val          690              - #   695              - #   700                          - - Tyr Ser Asn Pro Arg Gln Glu Thr Gly Ile Al - #a Gly His Pro Thr Tyr      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Gln Phe Ser Ala Asn Thr Gly Pro Ala His Ty - #r Met Thr Glu Gly        His                                                                                             725  - #               730  - #               735             - - Leu Thr Met Arg Gln Gly Ala Asp Arg Glu Gl - #u Ser Pro Met Thr Gly                  740      - #           745      - #           750                  - - Val Cys Val Gln Gln Ser Pro Val Ala Ser Se - #r                                  755          - #       760                                           __________________________________________________________________________

What is claimed is:
 1. A recombinant nucleic acid comprising a codingregion encoding a human tumor necrosis factor receptor associated factornumber two associated kinase protein comprising SEQ ID NO:2 or afragment thereof selected from the group consisting of residues 1-158,159-479 and 480-763 of SEQ ID NO:2, said coding region flanked by fewerthan 2 kb of native flanking sequence.
 2. The nucleic acid of claim 1,wherein said protein comprising SEQ ID NO:2.
 3. A method of making animmunogenic protein, comprising steps: introducing a nucleic acidaccording to claim 1 into a host cell, growing said host cell underconditions whereby said nucleic acid is expressed as a transcript andsaid transcript is expressed as a translation product comprising saidprotein, and isolating said protein from said cell.
 4. The method ofclaim 3, wherein said protein comprises SEQ ID NO:2.
 5. A recombinantnucleic acid comprising a sequence selected from the group consisting ofnucleotides 121-594, 595-1557 and 1558-2409, as set forth in SEQ IDNO:1, said sequence flanked by fewer than 2 kb of native flankingsequence.
 6. The nucleic acid of claim 5, comprising SEQ ID NO: 1.